Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (β£-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca 2Ψ permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7nitroquinoxaline-2,3-dione (CNQX). Cyclothiazide potentiated AMPA-and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2containing, Ca 2Ψ -impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.